The aim of the study was to replace mineral fertilizers with organic and biological fertilizers to improve nutrient uptake, plant growth and the concentrations of some important secondary metabolites in periwinkle (Catharanthus roseus L.). Periwinkle plants were grown under different rates of N supply (0, 20 and 40 mg kg−1 soil) and biological treatments (Azospirillum, Azotobacter, Azospirillum plus Azotobacter, Azospirillum plus fungal compost, Azotobacter plus fungal compost, and fungal compost). The concentrations of pigments and nutrients were measured by spectrophotometry and flame photometry. Secondary metabolites were analyzed by high-performance liquid chromatography (HPLC). Data were recorded for plant growth and development parameters, nutrient uptake and some secondary metabolites of periwinkle plants. The results showed that the N-fertilizer and biological treatments significantly improved most growth attributes and nutrient uptake and increased the concentrations of secondary metabolites as compared to the control. Maximum concentrations of root ajmalicine (0.54 mg g−1 DW), leaf vinblastine (0.96 mg g−1 DW) and root catharanthine (2.38 mg g−1 DW) were obtained from the treatment with Azospirillum under N-fertilizer at 20 and 40 mg kg−1 soil. Azotobacter along with fungal compost under N-fertilizer at 40 mg kg−1 soil induced the maximum concentration of leaf vindoline (1.94 mg g−1 DW). The highest concentration of root alkaloids (1.11 mg g−1 DW) was obtained from the treatment with compost under 40 mg N kg−1 soil. Azospirillum, Azotobacter and fungal compost combined with the N-fertilizer improved many morphological and nutrient characteristics. In conclusion, the growth and metabolism of C. roseus were significantly positively affected by the organic and biological fertilizers.