Nosocomial infections, in lay term known as hospital acquired infections, are caused mainly by airborne pathogens found in healthcare facilities and their surroundings. The aim of this study was to quantify and identify bacteria and fungi in a hospital, which is an understudied area of air quality in Sri Lanka. Air samples were collected in agar medium and petri plates containing sterile filter papers. The number of culturable and total airborne microorganisms was estimated by manual counting and fluorescent microscopy, respectively. The morphologically distant bacteria and fungi were identified by DNA sequencing. The statistical analysis revealed significant variances between studied sites ( < 0.05) where Outpatients Department and Respiratory Unit showed higher levels of airborne microbial load. Culturable microbial count was higher at noon (hospital visiting hours) compared to other sampling periods (after hospital visiting hours) within the hospital. Total count of airborne microbes was found to be the highest during the afternoon. The most sensitive zones such as Operating Theatre and Intensive Care Unit showed considerably higher counts of airborne microbes. Identification by molecular means revealed the presence of human pathogens in the hospital air including sp, sp, sp, sp, sp, sp, sp, sp, sp, sp, sp, and sp. In conclusion, the results from this study indicate that the hospital air was generally contaminated. Therefore, the implementation of proactive policies and strategies are needed to monitor hospital air quality in sensitive zones as well as other areas of the hospitals.