Isolation of Colletotrichum gloeosporioides (causal agent of yam anthracnose) from soil is difficult because of the presence of fast growing micro-organisms like Rhizoctonia spp., Rhizopus spp., Penicillium spp. and bacteria. Therefore, a semi-selective medium consisting of antimicrobial agents such as pencycuron 50 mg L−1, tolclofos-methyl 10 mg L−1, streptomycin sulphate 100 mg L−1, chloramphenicol 100 mg L−1 and chlortetracycline 100 mg L−1 was developed using potato dextrose agar as the basal medium. The procedure involved screening of candidate fungicides, developing a suitable combination of antimicrobial agents, assessing the proposed semi-selective medium and adjustment of pH of the medium to 5. The effectiveness of the medium is due to selective inhibition of micro-organisms by the antimicrobial agents and the development of distinct salmon-pink colonies of C. gloeosporioides.