Abstract Anal fistulae are said to arise from cryptoglandular infection of the anal glands, which lie within the intersphincteric space. The type and virulence of the micro-organism responsible may determine whether an analfistula develops. The microbiology of chronic anal fistulae has not been reported previously. Twenty-five consecutive anal fistulae were studied prospectively (eight intersphincteric fistulae, 12 trans-sphincteric fistulae, two suprasphincteric fistulae, one extrasphincteric fistula, one superficial fistula, one anovaginal fistula). There were 18 men and seven women, with a median age of 42 (range 22–71) years. Patients with Crohn's disease or acute anorectal suppuration were excluded. In 18 patients, 0.1 ml granulation tissue from the track of the fistula was obtained and processed within 4 h using standard microbiological techniques. Sixty-nine isolates representing at least 17 species were obtained. The predominant organisms were Escherichia coli (22 per cent), Enterococcus spp. (16 per cent) and Bacteroides fragilis (20 per cent). The majority of the growths were obtained only from enrichment. Bacteria from only one patient grew at a dilution of 103. Granulation tissue from 25 patients was processed for mycobacterial culture, and Mycobacterium tuberculosis was grown from one patient. No other mycobacterium was isolated. The chronic inflammation in anal fistulae does not seem to be maintained by either excessive numbers of organisms or organisms of an unusual type.