A hospital is an environment solely for diagnosing and treating patients. Contemporary research, however, reveals the possibility of users contracting diseases due to many factors, such as poor air quality. This research, therefore, delves into the critical indoor air quality assessment domain, focusing on selected units within the University of Cape Coast Hospital, Ghana. The study’s primary objective was to conduct a comprehensive microbial assessment of indoor air quality in eight different units (emergency room, operating theater, out-patient department, consulting rooms, laboratory, male ward, female ward, and ear, nose, and throat unit) of the hospital, shedding light on potential airborne bacteria present. Indoor and outdoor air were sampled using Koch’s sedimentation method. Colony forming units per cubic meter of air (cfu/m3) were determined with the Omeliansky formula. The bacteriological load within the units revealed that the out-patient department had the highest bacterial concentration (139.2±60.32×102 cfu/m3), immediately followed by Outdoor (135.1±43.63×102 cfu/m3), whereas ear, nose, and throat unit recorded the least concentration (0.4±0.57×102 cfu/m3). The remaining units range between 135.1±43.63×102 cfu/m3 and 0.4±0.57×102 cfu/m3. The morphological characteristics of the seven observed bacterial isolates (GSB 1-7) showed the presence of two cocci and five rods. Isolates 1 and 4 had a rhizoid form, isolates 2,3, and 5 had a circular form, while isolates 6 and 7 had filamentous forms. All isolates showed positive gram tests, and endospores were detected in isolates 1, 4, and 6. Bacterial isolates were identified as Bacillus mycoides, Micrococcus luteus, Staphylococcus epidermidis, Bacillus circulans, Staphylococcus saprophyticus, Bacillus subtilis, and Micrococcus sp. These outcomes indicate bacterial contaminations in the indoor environment, likely to pose a significant risk to patients, workers, and visitor’s safety. Therefore, rigorous monitoring and mitigation strategies are essential to ensure a safer environment in healthcare settings.