The study into the chemical contents and in-vitro antimicrobial activities of the methanolic leaf extract of Acanthospermum hispidum were carried out. The extract was evaluated for its antibacterial activity against four Gram positive (Staphylococcus aureus, Streptococcus pyogenes, Corynebacteria specie and Bacillus subtilis) and four Gram negative bacteria (Salmonella typhi, Klebsiella phumoniae, Pseudomonas aeruginosa and Escherichia coli). The fungal strains used were Aspergilus niger, Penicillium sp. and Candida albicans. The sensitive microorganisms (Conynebacteria sp., Pseudomonas aeragunosa, Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus) had zones of inhibition ranging from 12.20 ± 1.06 mm to 24.00 ± 1.00 mm at 100 mg/ml, while the standard drug (tetracycline 250 mg) had zones of inhibition in the range 20.27 ± 0.64 mm to 27.23 ± 0.68 mm against all the microorganisms tested in this study. The MIC/MBC against the tested organisms ranged from 25 mg/ml to 50 mg/ml and 50 mg/ml to 100 mg/ ml respectively. The results obtained from this study revealed that the leaf extract of Acanthospermum hispidum possesses antibacterial activity against some pathogenic microorganisms tested. The study confirmed the use of the leaf of Acanthospermum hispidum in some parts of Northern Nigeria as a remedy against diarrhoea, dysentery and other related diseases. In view of the reported uses of this plant, the present study investigated the antimicrobial effect of the methanol extract of A. hispidum on some Gram positive, Gram negative and some fungal species. Therefore, this study validates the medicinal use of Acanthospermum hispidum tradicinally in some parts of Northern Nigeria.