The purpose of this study was to prepare antimicrobial silk fibroin nanofibers from the aqueous formic acid solutions of silk fibroin and hydroxytyrosol with the in situ hydrolysis of oleouropein present in olive leaf extract using electrospinning method. With the use of aqueous formic acid solution of olive leaf extract and silk fibroin resulted in more uniform and beadless nanofibers. Morphological properties of electrospun nanofibers were investigated using Scanning Electron Microscope (SEM). The diameter of electrospun nanofibers ranged between 70 nm to 150 nm. The nanofiber diameter did not changed much with increasing concentration of olive leaf extract added into silk fibroin solution to be used in electrospinning process. The increase in olive leaf extract concentration resulted in beadless and uniform nanofiber structures. The average diameter of the nanofibers prepared with fibroin solution having 10 % olive leaf extract was determined as 85 ± 10 nm. Results revealing the formation of smoother and uniform nanofibers was attributed to the crosslinking effect of oleuropein and polyphenols present in olive leaf extract with certain functional groups in silk fibroin structure. Antibacterial properties of hydroxytyrosol loaded nanofibers against Staphylococcus epidermidis (Gram +) and Escherichia coli (Gram -) were confirmed with the clear inhibition zones observed in disc diffusion tests. Silk Fibroin nanofibers loaded with hydroxytyrosol may offer a new alternative biomaterial to be used in wound dressing or medical textile applications.