The COVID-19 outbreak was a serious challenge for countries around the globe. With the objective of mitigating the spread of the virus, both national and international health organizations swiftly enacted quarantine measures across numerous cities around the globe. This presented a unique chance to evaluate the consequences of human actions on the quality of the air. This study aimed to investigate airborne microbial levels in different outdoor locations in Al-Madinah City, Kingdom of Saudi Arabia (KSA), during the COVID-19 pandemic by comparing lockdown against non-lockdown conditions. Twelve outdoor locations were investigated in terms of microbial total counts using the open plate method during and after the COVID-19 lockdown. Environmental factors that could affect the airborne microbial load, including humidity, temperature and wind speed, were recorded during the assessment. The means of the total colony-forming units (CFU) for each cubic meter (m³) of air were calculated. Lockdown restrictions caused significant decreases in the biological contaminants in all locations compared with the numbers after the pandemic. Gram-positive bacteria represented most of the samples, with fewer fungal strains detected. The outdoor average total bacterial counts ranged between 0.00±0.00-8337.50±248.98 CFU/m³, compared with 2903.75±407.60-19722.50±475.03 CFU/m³ after the pandemic. The mean concentrations of total fungi were lower than those of bacteria and ranged between 0.00±0.00-143.75±131.75 CFU/m³ during the COVID-19 lockdown and were elevated after the lockdown to reach 28.75±49.80-776.25±298.78 CFU/m³. Based on the available data, there are no studies comparing outdoor microbial counts during and after the COVID-19 pandemic. Therefore, this research offers additional perspectives on the air quality experienced amidst the COVID-19 pandemic and the subsequent implementation of lockdown measures and could serve as a valuable resource for monitoring and implementing measures to control air pollution.