In 1867 she joined the Society of St Margaret at East Grinstead, Sussex, one of the earliest religious orders for women in the post-Reformation Church of England.

[3] From 1879 to 1884 Macfarlane was Matron of the Servants Training Institute, a new Church of England initiative in East Melbourne designed to prepare girls for domestic service.

On 8 February 1884 Macfarlane was ordained to the "Female Diaconate" by James Moorhouse, the Anglican Bishop of Melbourne, at Christ Church, South Yarra.

She initially worked with H. F. Tucker, the Vicar of Christ Church South Yarra, and appears later to have lived at Bishopscourt, East Melbourne, with Bishop and Mrs Moorhouse.

[2] Peter Sherlock argues that since Macfarlane was ordained with the episcopal laying on of hands, with Bishop Moorhouse intending to create a "Female Diaconate", she should be considered to be the first woman to be a member of the clergy in the Anglican Church in Australia.