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<i>Staphylococcus lugdunensis</i> is a coagulase-negative-staphylococci (CoNS) that lately has gained special attention in public health as a human pathogen and also as a bacteriocin-producer bacteria. In this study, we characterized 56 <i>S. lugdunensis</i> isolates recovered from human samples in two Spanish hospitals. Antimicrobial susceptibility testing was performed and antimicrobial resistance and virulence genotypes were determined. Antimicrobial activity (AA) production was evaluated by the <i>spot-on-lawn</i> method against 37 indicator bacteria, including multidrug-resistant (MDR) isolates, and the presence of the <i>lug</i>D gene coding for lugdunin bacteriocin was analyzed by PCR. The antibiotic resistance detected was as follows (% resistance/genes detected): penicillin (44.6%/<i>bla</i>Z), oxacillin (1.8%/<i>mecA</i> on SCC<i>mec</i>-V), erythromycin-clindamycin inducible (7.1%/<i>erm</i>(C), <i>msrA</i>), tetracycline (5.3%/<i>tetK</i>), gentamicin and/or tobramycin (3.6%/<i>ant</i>(4′)-Ia, <i>acc</i>(6′)-<i>aph</i>(2″)), and fosfomycin (21.4%). A MDR phenotype was detected in 5% of isolates. Twenty-one of the <i>S. lugdunensis</i> isolates showed susceptibility to all 20 antibiotics tested (37.5%). The screening for AA revealed 23 antimicrobial producer (AP) isolates with relevant inhibition against coagulase-positive-staphylococci (CoPS), including both methicillin-susceptible and –resistant <i>S. aureus</i>. The <i>lug</i>D gene was detected in 84% of the 56 <i>S. lugdunensis</i> isolates. All of the AP <i>S. lugdunensis</i> isolates (<i>n</i> = 23) carried the <i>lug</i>D gene and it was also detected in 24 of the non-AP isolates, suggesting different gene expression levels. One of the AP isolates stood out due to its high antimicrobial activity against more than 70% of the indicator bacteria tested, so it will be further characterized at genomic and proteomic level.