Knowledge of the microbial diversity on hospital bed linens has implicit significance for infection control because bed linens are likely reservoir and vehicles for healthcare-associated Infections (HAIs). In this study, the diversity of microorganisms that persist on bed linens at Benue State University Teaching Hospital (BSUTH), Makurdi, Nigeria was evaluated. Patients’ bed and surface areas on bed linens were chosen at random from ten different wards and the swab sampling method employed. Of the 30-bed linens sampled, bacteria were more prevalent at 63.3%, and fungi at 34.48%. We identified both bacteria and fungi from eight wards, only fungi from the antenatal ward and no contamination from the amenity ward. Staphylococcus aureus, Escherichia coli, Klebsiella, and coagulase-negative Staphylococcus dominated the bacterial profile. Similarly, Aspergillus niger, Candida albicans, and Microsporium ferrugenium fungi were also recovered from the bed linens. Amongst the bacteria identified, Escherichia coli gave the highest prevalence at 36.84%, whereas, Candida albicans revealed the highest among the fungi isolates with 50%. Chi-square analysis showed a significant relationship (χ2 = 68.48, df =8, P<0.05) between the microbial contamination and the wards sampled. The mean bacterial count (cfu/ml) was checked for each ward and the female surgical ward showed the highest count of 31.3 × 103 and the paediatric ward had the least count of 2.3 × 103. Control beddings were also contaminated with 1.3 × 103. These results suggest that the bed linens from the hospital wards were contaminated with pathogenic microbes which can contribute to HAIs.