Objectives Psychoactive substance experimentation among Tunisian adolescents remains one of the most threatening public health concerns. In spite of this, little is known about the prevalence and predictors of these behaviors in the Tunisian context. This study aims to assess the prevalence of tobacco, alcohol and illicit drug experimentation and its predictive factors among adolescents in the delegation of Nfidha, Sousse governorate, Tunisia. Methods We conducted a cross sectional study in middle and high schools in the delegation of Nfidha, Sousse governorate, Tunisia, during the first trimester of the 2019–2020 school year using an anonymous questionnaire self-administered to a randomly drawn representative sample of 1,352 pupils. Data collected included socio-demographic characteristics, substance experimentation among school-adolescents, academic performance as well as family and peer’ factors. Results Tobacco was the most commonly experimented substance with a prevalence rate of 17.5 % (CI 95 %: 14.8–18.8 %). The prevalence of alcohol and illicit drugs use were 3.3 % [CI 95 %: (2.1–3.9 %)] and 2 % [CI 95 %: (1.1–2.6 %)] respectively. Being male (Adjusted (AOR)=4.09, p<0.001), being a high school pupil (AOR=2.81, p<0.001), having experiencing academic failure (AOR=1.60, p=0.007) and having a father and siblings who use tobacco (AOR=1.72, p=0.002; AOR=2.03, p=0.001 respectively) were the predictors of tobacco experimentation in the multivariate logistic regression analysis. Additionally, being male (AOR=8.40, p<0.001), having experienced academic failure (AOR=2.76, p=0.017) as well as having a father and siblings who use alcohol (AOR=5.90, p<0.001; AOR=3.03, p=0.045 respectively) and having experienced tobacco (AOR=4.28, p<0.001) were the determinants of alcohol experimentation. Similarly, having a history of academic failure (AOR=3.44, p=0.041), having peers who use illicit drugs (AOR=3.22, p=0.028), having a history of tobacco and alcohol experimentation (AOR=6.52, p<0.001; AOR=3.49, p=0.046 respectively) were the predictors of illicit drug experimentation among adolescents. Conclusions A substantial number of socio-demographic and environmental factors have been identified as being involved in experimentation with psychoactive substance during adolescence. Therefore, further prevention programs targeting not only adolescents, but also all risk factors are needed.