Antimicrobial resistance (AMR) is a global threat that has led to nearly incurable ailments, with prolonged disease occurrences being linked to cancer development. Fungal endophytes from medicinal plants produce diverse natural compounds that can combat the progression of AMR pathogens. Kirkia acuminata is an overharvested African medicinal plant that is used to treat various ailments, such as diarrhoea, toothache and thrush; however, there is little knowledge on the biological potential of its fungal endophytes. Thus, the current study aimed to assess the antimicrobial, cytotoxicity and anticancer properties of fungal endophytes associated with K. acuminata stems. Culturable fungal endophytes from surface-sterilised stems of K. acuminata were identified through sequencing of the internal transcribed spacer (ITS) region. Ethyl acetate extracts of the isolates were assessed for their antimicrobial activity using the broth microdilution assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cytotoxic profiles of the crude extracts against non-cancerous human embryonic kidney (HEK-293) cells. Moreover, the alamarBlue assay was used to screen the anticancer activity of the non-cytotoxic extracts against cervical cancer ME-180 and melanoma A375 cancerous cells. A total of 18 morphologically distinct isolates distributed among the genera Diaporthe, Neofusicoccum and Pseudofusicoccum were obtained. Seventeen out of the eighteen isolates possessed inhibitory activity against Candida albicans, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, with overall MIC values between 0.31 and 2.5 mg/mL. However, only four isolates, D. macadamiae KaS-4, D. neotheicola KaS-6, N. parvum KaS-3 and P. olivaceum KaS-5, were non-cytotoxic against the non-cancerous HEK-293 cells and exhibited moderate inhibitory activity against the ME-180 and A375 cancerous cells. Thus, this study highlights the potential of fungal endophytes from K. acuminata in producing safe natural compounds with antimicrobial and anticancer action for drug discovery.