In recent decades, pathogens have become resistant towards conventionally available antibiotics. This phenomenon persistently proliferates while increasing the number of individuals suffering from diseases and morbidity. In consequence, a pressing need arises for more effective antimicrobial agent to inhibit these infectious pathogens. One specific biomaterial of interest known as riboflavin or vitamin B2 has been shown to exhibit antimicrobial properties when activated with ultraviolet A (UVA) irradiation. The antimicrobial efficacy of riboflavin without the need for irradiation using UVA exposure for the microbial isolates tested was shown for the first time in this study. Furthermore, previous treatments with standard drugs and then with riboflavin application significantly inhibited the growth of the selected pathogens. This combinational or stand-alone riboflavin application could be further explored for enhanced management of blood borne pathogens.