Background: In 2012 we undertook a screening study for Enterobacteriaceae with extended-spectrum β-lactamases (ESBLs), derepressed or acquired high-level AmpC cephalosporinases, and metallo-β-lactamases (MBLs), and also methicillin-resistant Staphylococcus aureus (MRSA), in a long-term care facility (LTCF1) and the associated acute care hospital geriatric ward in Bolzano, northern Italy. The study followed up an initial survey carried out in LTCF1 in 2008. For comparison, screening in 2012 was extended to a second LTCF. Methods: Urine samples and rectal, inguinal, oropharyngeal, and nasal swabs were plated on selective agars. Isolates were typed by pulsed-field gel electrophoresis. Resistance genes and Escherichia coli belonging to ST131 were sought by PCR. Demographic data were collected. Results: Fewer residents of LTCF1 were colonized with multidrug-resistant (MDR) bacteria in 2012: all MDR organisms, 53.8% vs 74.8% in 2008; ESBL producers, 49.0% vs 64.0% in 2008; MRSA, 13.2% vs 38.7% in 2008; only 2 MBL-producers were isolated in 2012 vs 8 in 2008. Colonization of staff in LTCF1 by MDR bacteria had also decreased (overall 10.5% in 2012 vs 27.5% in 2008). Changed case mixes and risk factors, together with strengthened hygiene measures probably underlie the changes. Colonization proportions in 2012 in LTCF2 were similar to those in LTCF1. By contrast there was no significant change in the proportion of patients colonized by MDR bacteria in the geriatric ward (22.2% in 2008 vs 22.7% in 2012). Conclusions: A significant decrease in the proportions of staff and residents of an LTCF colonized by MDR bacteria was observed over a 4-y interval.