Failure of antibiotic treatment in eradication of bacterial tonsillitis induced us to postulate presence of ''bacterial biofilms'' attached to living tissues that can be considered as an etiologic factor, among others. The knowledge about biofilm existence supports a new concept to explain chronic infections. Tonsillectomy is often the required choice as a consequence of frequent tonsillitis during the previous years, subsequently a novel studies propose using of non-antibiotic and non-surgical modalities for eradication of biofilm-related tonsillitis. In our designed study we used ''vinegar'' as anti-biofilm agent due to its antimicrobial effect. Vinegar is a sour liquid composed mainly of acetic acid, which has an important role in disruption of biofilm aggregations. This study has been undertaken to determine biofilm production ability by Streptococcus pyogenes isolated from tonsillitis patients and to evaluate effectiveness of different types of vinegar for eradication of Streptococcal-tonsillar biofilm, in vitro. Twenty nine isolates of S. pyogenes belonged to 19 patients, were tested for biofilm production by using Microtiter-plate method. Also, Modified Microtiter-plate method was conducted to study the effect of different vinegar types (Date, Apple, and Grape) as anti-biofilm, in vitro. Out of 75.9% produced biofilm according to depending biofilm criteria, while 24.1% were non-biofilm producers. Biofilm producer isolates distributed into 12(54.5%) and 10(45.5%) which was detected on tonsil surface and crypts respectively. Concerning the anti-biofilm activity, our results demonstrated that types of vinegar eradicated biofilm by (100%), (95.5%), and (90.9%) for Date, Apple, and Grape vinegar respectively in compare with distilled water as negative control. It was concluded that there was no significant differences in biofilm production between S. pyogenes isolated from tonsil surface and crypts. Furthermore, our study concluded that the three types of vinegar eradicated streptococcal biofilm remarkably, but Date vinegar was the best for eradication of streptococcal biofilm in vitro.