Al-Madinah Al-Munawwarah is the second holiest site in Islam. The possibility of new emerging microbes is valid due to the increased number of pilgrims. The objectives of the current study were to estimate the numbers of fungi and bacteria inside and outside Al-Haram Al-Nabawi and to find whether new bacterial and fungal species have emerged compared to previous studies. Air samples were collected twice a day from 12 spots and four directions during the pilgrim year of 2013 for four consecutive weeks by using the sedimentation method. Thirty five genera and fifty eight species were identified. The most recovered bacterial genera were Staphylococcus, Micrococcus, Bacillus, and Dermacoccus with 32.47%, 18.18%, 12.85%, and 11.23%, respectively. Fifty nine isolates of fungi were molecularly identified. Aspergillus species had the highest percentage (78%). The other fungal genera identified (, ,, ,, ,, , and) had less than 5% frequency. In places such as Al-Haram Al-Nabawi, a large and crowded public (millions) exist especially during pilgrimages and Ramadan, thus, exposure to microorganisms is high. On the other hand, microorganism infectivity depends on many factors including their virulence, landing site, and person's immunity. For those reasons, many aspects should be considered to avoid aerosol contaminants.