The present research study evaluated the effects of four concentrations of Benzylaminopurine (BAP) in combination with three concentrations of Naphthalene acetic acid (NAA), as well as methyl jasmonate and nano-sized TiO2 elicitors on callus induction, pharmaceutical important flavones and antioxidant compositions in leaf-derived calli of Teucrium polium. The feed-forward Artificial Neural Network (ANN) model, as well, was applied to predict the effects of hormones and elicitors on phenolic compounds production. The best callus induction was obtained with basal media fortified with 1.5 mg L− 1 BAP plus 0.5 mg L− 1 NAA, followed by 1.5 mg L− 1 BAP. Based on biochemical features (the total content of phenols, Ortho-diphenols, flavonoid, flavone, and rosmarinic acid), the extraction has substantially by elicitation raised in comparison to control, with the maximum ones in calli elicited with methyl jasmonate (50 µM). The exception was phenolic acids possessed much greater content in 10 mg L− 1 nano TiO2-treated calli. The high antioxidant activities (IC50 2.066 ± 0.24 µg mL− 1 and 383.79 ± 33.60 mg Fe+ 2 100 g− 1 DW) with approximate 2- and 1.5-fold increases compared to control, was observed in hydromethanolic extracts of calli elicited by 50 µM methyl jasmonate on an MS medium containing 1.5 mg L− 1 BAP plus 0.5 mg L− 1 NAA. Likewise, the highest contents of anti-genotoxic flavones apigenin and luteolin were measured at pretreatment of callus with 50 µM methyl jasmonate on the media supplemented with 1 mg L− 1 BAP (180.67 ± 2.52 and 201.11 ± 3.63 µg 100 g− 1 extract, respectively) and 1.5 mg L− 1 BAP plus 0.5 mg L− 1 NAA (206.01 ± 7 and 256.22 ± 2.1 µg 100 g− 1 extract, respectively). ANN with hyperbolic-tangent (10-1-3) activation function, R2 0.99, SSE 0.01, and RE 0.001 showed a strong correlation between ANN model outputs and the experimental amounts of polyphenols, Ortho-diphenols, and phenolic acids. The results support that methyl jasmonate elicitation could more trigger and increase the pharmaceutical phenolic compositions in T. polium callus cultures as compared with nano-sized TiO2 treatment. Meanwhile, the ANN model could successfully interconnect the output parameters (phenolic metabolites) to input variables (hormone and elicitor).