The growing number of patient morbidity related to nosocomial infections has placed an importance on the development of new antibacterial coatings for medical devices. Here, we utilize the versatile adhesion property of polydopamine (pDA) to design an antibacterial coating that possesses low-fouling and nitric oxide (NO)-releasing capabilities. To demonstrate this, glass substrates were functionalized with pDA via immersion in alkaline aqueous solution containing dopamine, followed by grafting of low-fouling polymer (poly(ethylene glycol) (PEG)) via Michael addition and subsequent formation of N-diazeniumdiolate functionalities (NO precursors) by purging with NO gas. X-ray photoelectron spectroscopy confirmed the successful grafting of PEG and formation of N-diazeniumdiolate on polydopamine-coated substrates. NO release from the coating was observed over 2 days, and NO loading is tunable by the pDA film thickness. The antibacterial efficiency of the coatings was assessed using Gram-negative Pseudomonas aeruginosa (i.e., wild-type PAO1 and multidrug-resistant PA37) and Gram-positive Staphylococcus aureus (ATCC 29213). The NO-releasing PEGylated pDA film inhibited biofilm attachment by 96 and 70% after exposure to bacterial culture solution for 24 and 36 h, respectively. In contrast, films that do not contain NO failed to prevent biofilm formation on the surfaces at these time points. Furthermore, this coating also showed 99.9, 97, and 99% killing efficiencies against surface-attached PAO1, PA37, and S. aureus bacteria. Overall, the combination of low-fouling PEG and antibacterial activity of NO in pDA films makes this coating a potential therapeutic option to inhibit biofilm formation on medical devices.