Colibacillosis is the most common bacterial disease in the poultry industry. The isolation of Escherichia coli (E. coli) strains with multiple resistance to various classes of antimicrobials has been increasing in recent years. In this study, antimicrobial resistance features, serotyping and the presence of avian pathogenic Escherichia coli (APEC) virulence genes were investigated on a total of 71 E. coli strains isolated during outbreaks of colibacillosis in laying hens. The correlation between these features was evaluated. The most frequently isolated serogroups were O2 and O88. Resistance was often detected with nalidixic acid (49%) and ampicillin (38%), while all strains were sensitive to ceftiofur and florfenicol. Overall, 25% of the isolates showed resistance to at least three or more antimicrobial classes (multidrug-resistant strains), and 56% of the isolates were defined as APEC strains (due to the presence of at least five virulence genes). Correlation between the different parameters (virulence genes, serogroup and antimicrobial resistance) did not reveal relevant associations. The comparison of the obtained results with those of similar studies highlighted the importance of continuous monitoring in order to have a better understanding of colibacillosis. An evaluation of the national epidemiological situation would allow, especially with regard to antimicrobial resistance, to focus on the right measures in order to prioritize the available resources for effective disease control.