Despite the existing antibiotics, antimicrobial resistance is a major challenge. Consequently, the development of new drugs remains in great demand. Quinones is part of a broad group of molecules that present antibacterial activity besides other biological properties. The main purpose of this study was to evaluate the antibiofilm activities of synthetic N,O-acetals derived from 2-amino-1,4-naphthoquinone [7a: 2-(methoxymethyl)-amino-1,4-naphthoquinone; 7b: 2-(ethoxymethyl)-amino-1,4-naphthoquinone; and 7c: 2-(propynyloxymethyl)-amino-1,4-naphthoquinone] against methicillin-resistant Staphylococcus aureus (MRSA). The derivatives 7b and 7c, specially 7b, caused strong impact on biofilm accumulation. This inhibition was linked to decreased expression of the genes fnbA, spa, hla and psmα3. More importantly, this downregulation was paralleled by the modulation of global virulence regulators. The substitution of 2-ethoxymethyl (7b) in comparison with 2-propynyloxymethyl (7c) enhanced sarA-agr inhibition, decreased fnbA transcripts (positively regulated by sarA) and strongly impaired biofilm accumulation. Indeed, 7b triggered intensive autolysis and was able to eliminate vancomycin-persistent cells. Consequently, 7b is a promising molecule displaying not only antimicrobial effects, but also antibiofilm and antipersistence activities. Therefore, 7b is a good candidate for further studies involving the development of novel and more rational antimicrobials able to act in chronic and recalcitrant infections, associated with biofilm formation.