An in-house portable, economical, platform-independent Ultraviolet Germicidal Irradiation (UVGI) system was developed for the effective sterilization of bacteria and fungi using a wireless mode of operation. Microbiological validation of the system was performed using biofilm-forming gram-positive spore-forming bacteria Bacillus subtilis, gram-negative bacteria Escherichia coli, and the fungal strain Candida albicans M-207, a highly biofilm-forming multi-drug resistant clinical yeast isolate. The viability of the cultures exposed to UVGI was evaluated by Colony Forming Unit count and the MTT assay. Morphological studies of the ultraviolet (UV)-irradiated cultures were performed using Scanning Electron Microscopy. UVGI surface sterilization was effective in the sterilization of bacteria and yeast during 15 min of exposure at a distance of 1 m with an average UV intensity of 0.694 mWcm-2. A linear relationship was observed between distance and time of exposure to UVGI. Therefore, this pilot study validated that the newly developed UVGI surface sterilizer is effective in the disinfection and sterilization of biofilm-forming bacterial and fungal pathogens. They can be scaled for wider cost-effective use globally in larger settings of hospitals and other public spaces where the pathogen infection transmission potential is high.