The aim of this study was to characterize a broad collection of isolates of Listeria monocytogenes, of different serotypes, recovered in Portugal between 2003 and 2007 from foods (n=353) and from clinical cases of human listeriosis (n=95), in terms of antimicrobial susceptibility. All the isolates were susceptible to ampicillin, the preferred agent to treat listeriosis. Resistances to nitrofurantoin (n=99), to ciprofloxacin (n=18), to erythromycin (n=10), to tetracycline (n=2), to gentamicin (n=1) and to rifampicin (n=1) were observed. One hundred (28.3%) and 20 (21.0%) food and clinical isolates, respectively, were resistant to at least one antibiotic. Eight isolates (1.8%) were resistant to two or more antimicrobials of different classes, and all were collected from foods. Serogroup IVb included the highest percentage of isolates resistant to erythromycin. The highest percentages of isolates resistant to nitrofurantoin were of serogroup IVb and IIc. It was demonstrated that the incidence of antibiotic-resistant isolates of L. monocytogenes, during the period 2003 to 2007, was low in Portugal but still higher than that observed in other countries. Given the increasing population at greater risk of listeriosis, namely, the elderly, the high mortality rate of the infection and the detection of resistant isolates, monitoring for antibiotic resistance in strains of L. monocytogenes on a large scale, and assessing the risk of infection by these strains, is highly recommended.