In can be found in habitats modified by humans, such as gardens, agroecosystems, hedgerows, lawns, quarries, urban green spaces, walls and bridges.

[5] In agricultural settings, P. opilio is common in temperate cropland, living among crops such as corn, alfalfa, small grains, potatoes, cabbage, strawberries, and apple.

[6] P. opilio apparently prefer vertical surfaces, including tree trunks and vertical man-made structures like fences and walls,[4] with a preference for wood substrate due to its low thermal conductivity and slow release of moisture, being a thermophilous and moderately hygrophilous species.

Males tend to have smaller bodies than females, but have noticeably larger pedipalps and chelicerae with prominent outgrowths (horns) on the dorsal side of the second segment.

Males can be differentiated from females by their longer pedipalps and the existence of long, forward-pointing processes, appearing similar to horns, on the second segment of their chelicerae.

Their tarsus, the segment of their legs furthest from their body, have numerous pseudosegments called tarsomeres that make them prehensile, enabling P. opilio to use them in climbing, such as by curling them around twigs; courtship of mates; and male—male combat.

P. opilio's eggs are smooth, spherical in shape, and about 0.55 mm in diameter, laid in clusters ranging from ten to several hundred.

[12] Most of their time is spent stationary, but P. opilio have also been observed walking and leg palpating—making tapping movements with their sensory legs—as well as drinking, feeding, and grooming.

[7] P. opilio is a generalist predator and scavenger that feeds on soft-bodied animals found in crops, such as aphids, caterpillars, leafhoppers, beetle larvae, and mites.

Two or more generations may occur within a year in some areas of North America, in which case eggs, immatures, and adults may all overwinter.

The mating season of P. opilio is three months long, as examined in the cold humid "Dfb" climate subcategory of the Köppen–Geiger map.

[11] When observed in small, fenced-in areas of soybean fields, P. opilio nymphs of different sizes and adults of different sexes inhabited different vertical levels, moving between the ground and the bottom, middle, and top of plants at different times of the day.

Adult males tend to remain on the ground by 03:00, spending more time there than females and large nymphs that return to the plants by then.

Foundational work on the developmental biology of this species was established by Manfred Moritz and Dietrich Winkler in the 20th century, and resurrected by Prashant Sharma.

[15] Further investigation of homeobox genes like homothorax, labial, Deformed, and Sex combs reduced revealed the Hox code logic that specifies each appendage identity in the head of arachnids.

Guilherme Gainett et al. identified and examined the function of the genes Deformed (Dfd) and Sex combs reduced (Scr).

Knockdown of another gene, Epidermal growth factor receptor (Egfr) also caused shortened legs and the loss of tarsomeres.

[18] Genomic resources established for P. opilio have shown that Opiliones are in fact not closely related to scorpions, as had long been suggested based on morphology; scorpions are understood to be more closely related to spiders (as part of the clade Arachnopulmonata) on the basis of a shared whole genome duplication that excludes groups like Opiliones, mites, and ticks.