The rapid rise in antimicrobial resistance in bacteria has generated an increased demand for the development of novel therapies to treat contemporary infections, especially those caused by methicillin-resistant Staphylococcus aureus (MRSA). However, antimicrobial development has been largely abandoned by the pharmaceutical industry. We recently isolated the previously described thiopeptide antibiotic nosiheptide from a marine actinomycete strain and evaluated its activity against contemporary clinically relevant bacterial pathogens. Nosiheptide exhibited extremely potent activity against all contemporary MRSA strains tested including multiple drug-resistant clinical isolates, with MIC values 0.25 mg l(-1). Nosiheptide was also highly active against Enterococcus spp. and the contemporary hypervirulent BI/NAP1/027 strain of Clostridium difficile but was inactive against most Gram-negative strains tested. Time-kill analysis revealed nosiheptide to be rapidly bactericidal against MRSA in a concentration- and time-dependent manner, with a nearly 2-log kill noted at 6 h at 10 × MIC. Furthermore, nosiheptide was found to be non-cytotoxic against mammalian cells at >>100 × MIC, and its anti-MRSA activity was not inhibited by 20% human serum. Notably, nosiheptide exhibited a significantly prolonged post-antibiotic effect against both healthcare- and community-associated MRSA compared with vancomycin. Nosiheptide also demonstrated in vivo activity in a murine model of MRSA infection, and therefore represents a promising antibiotic for the treatment of serious infections caused by contemporary strains of MRSA.