Invasive Haemophilus influenzae (Hi) disease poses a significant global health challenge. With the relaxation of COVID-19 pandemic measures and declining H. influenzae serotype b (Hib) vaccination coverage, there is concern about a potential increase in Hi cases worldwide. This study analyzed 1437 invasive Hi isolates in Brazil over 13years, determining capsular serotypes, antimicrobial susceptibility, and genetic relatedness through multilocus sequence typing. The primary source of isolation for these invasive H. influenzae isolates was blood (54.4%), followed by cerebrospinal fluid (37.1%) and lung specimens (8.5%), respectively. Consequently, bacteremia (47%) was the most common clinical presentation, followed by meningitis (39.6%) and pneumonia (13.4%). Non-encapsulated Hi (NTHi) predominated among the isolates (51.4%), along with serotype a (22%) and serotype b (21.5%) among the encapsulated isolates. The majority of the encapsulated isolates were isolated from children under 14years of age (76.7%), while NTHi isolates were identified in patients older than 15years, particularly those ≥ 60years old (40%). Ampicillin resistance was observed in 17.1% of cases, displaying β-lactamase production as the principal resistance mechanism. MLST revealed a diverse NTHi population, whereas the encapsulated isolates presented a clonal structure. This study describes the prevalence of NTHi isolates circulating in Brazil after two decades of the Hib vaccine immunization program. Continuous universal surveillance is crucial for implementing prompt public health measures to prevent and control invasive Hi disease and monitor changes in antibiotic resistance profiles.