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<i>Haemophilus influenzae</i> invasive disease is a severe infection that needs rapid antibiotic therapy. The aim of the study was to perform and evaluate the serotype distribution, antibiotic susceptibility and molecular characteristics of 392 <i>H. influenzae</i> invasive isolates collected during 2017–2021 in Italy. The majority of isolates were NTHi (305/392, 77.8%), followed by Hib (49/392, 12.5%). Ampicillin resistance was frequently detected (85/392, 21.7%): 12.2% were β-lactamase producers (all <i>bla</i>_TEM except one <i>bla</i>_ROB), 9.4% were β-lactamase-negative ampicillin-resistant (BLNAR), with mutations in the <i>fts</i>I gene. Six isolates were resistant to ciprofloxacin, with substitutions in GyrA and ParC. An MLST analysis revealed the occurrence of international resistant clones, such as ST103 and ST14, highlighting the importance of molecular surveillance.