This fish, like most other darter species, tends to inhabit clear, rocky riffles of streams, creeks, and small rivers.

[4] Etheostoma rufilineatum exhibits typical darter behavior, preferring to live in riffles of small to medium-sized creeks, streams, and rivers, and is rarely if ever found in pools.

This fish is also known to inhabit shallow water shoals over bedrock, as long as some scattered cobble or gravel is available.

However, living in shallower water may make this darter more susceptible to predation from terrestrial hunters, such as wading birds and raccoons (Procyon lotor).

This fish feeds on aquatic and terrestrial insect larvae and other small invertebrates such as midgeflies, black flies, caddisflies, and water mites.

[6] These invertebrate species tend to be more abundant in areas inhabited by the redline darter, so are readily available as a food source.

Availability of different food sources varies by season and location; the feeding habits of redline darters change accordingly.

Increased pollution and siltation can result in higher water turbidity, making it difficult for this darter, and many other fish species, to forage for food, breed, and potentially decreasing the ability to avoid predators.