In this work, a multilayer antibacterial and antifungal superhydrophilic film was prepared on the 316L stainless steel surface by mussel-inspired depositions of polydopamine (PDA) and silver nanoparticles (Ag NPs) followed by modification with methoxy-polyethylene-glycol thiol (mPEG-SH, MW5000). The results of the scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) indicated that the critical hierarchical micro/nanostructures were mainly constructed by the combination of PDA and Ag NPs. After modified with mPEG-SH, the resulting surface showed excellent superhydrophilicity with a water contact angle of 0 degrees. Further, the as-prepared superhydrophilic surfaces exhibited enhanced and more durable antibacterial performances against the Gram-negative Escherichia coli and the Gram-positive Staphylococcus aureus under immersed environments. More importantly, the superhydrophilic surfaces also showed excellent antifungal activities against the fungi Penicillium F2-1 under atmospheric environments. The enhanced antimicrobial properties were resulted from the synergistic effects of anti-adhesion property of absorbed water layers, bactericidal property of Ag NPs and stereo hindrance effect of mPEG-SH molecular chains.