BackgroundThe vast majority of patients with acute tonsillitis (AT) are managed in general practice. However, occasionally patients are referred to hospital for specialized management because of aggravated symptoms and/or findings suggestive of peritonsillar involvement. No prospective studies have been conducted aiming to investigate the prevalent and significant microorganisms in this highly selected group of patients. We aimed to describe the microbiological findings of acute tonsillitis with or without peritonsillar phlegmon (PP) in patients referred for hospital treatment and to point out potential pathogens using the following principles to suggest pathogenic significance: (1) higher prevalence in patients compared to healthy controls, (2) higher abundance in patients compared to controls, and (3) higher prevalence at time of infection compared to time of follow up.MethodsMeticulous and comprehensive cultures were performed on tonsillar swabs from 64 patients with AT with (n = 25) or without (n = 39) PP and 55 healthy controls, who were prospectively enrolled at two Danish Ear-Nose-Throat Departments between June 2016 and December 2019.ResultsStreptococcus pyogeneswas significantly more prevalent in patients (27%) compared to controls (4%) (p< 0.001). Higher abundance was found in patients compared to controls forFusobacterium necrophorum(mean 2.4 vs. 1.4, p = 0.017) andS. pyogenes(mean 3.1 vs. 2.0, p = 0.045) in semi-quantitative cultures.S. pyogenes, Streptococcus dysgalactiae, and Prevotellaspecies were significantly more prevalent at time of infection compared to follow up (p= 0.016,p= 0.016, andp= 0.039, respectively). A number of species were detected significantly less frequently in patients compared to controls and the mean number of species was significantly lower in patients compared to controls (6.5 vs. 8.3, p < 0.001).ConclusionsDisregardingPrevotellaspp. because of the prevalence in healthy controls (100%), our findings suggest thatS. pyogenes,F. necrophorum, andS. dysgalactiaeare significant pathogens in severe AT with or without PP. In addition, infections were associated with reduced diversity (dysbacteriosis).Trial registrationThe study is registered in the ClinicalTrials.gov protocol database (# 52,683). The study was approved by the Ethical Committee at Aarhus County (# 1-10-72-71-16) and by the Danish Data Protection Agency (# 1-16-02-65-16).Supplementary InformationThe online version contains supplementary material available at 10.1186/s12879-023-08420-8.