Fusobacterium necrophorum (FN) is the predominant pathogen in peritonsillar abscesses, which is a relatively frequent complication of acute tonsillitis. The study aimed to explore if FN is a significant pathogen in acute tonsillitis, examine the prevalence of FN in acute tonsillitis patients, and describe the clinical and biochemical characteristics of FN-positive patients. A 6-month prospective study was conducted in a Danish general practice with eight physicians. One hundred acute tonsillitis patients and 100 healthy controls aged 15-40 years were included in the study. The prevalence of FN was (non-significantly) higher among acute tonsillitis patients (16 %) compared to healthy individuals (9 %) (P = 0.199). This trend was border significant for patients aged 15-29 years (24 vs 9 %) (P = 0.050). Significantly, more FN-positive patients were men (75 %) compared to patients growing other bacteria (17 %) or mixed oral flora (27 %) (P < 0.001). Centor scores, individual clinical symptoms, and infection markers were similar between patient growing FN and mixed oral flora. FN is possibly a significant and prevalent pathogen in acute tonsillitis among teenagers and young adults. Patients with FN-positive acute tonsillitis do not seem to be more clinically or biochemically affected than patients without growth of bacterial pathogens.