Canola (Brassica napus L.) is a very important agricultural and industrial crop. Hence, the effects of chemical and biological treatments on canola oil production and nutrient uptake, under calcareous conditions, were evaluated in a field experiment. Phosphorus (P) fertilizer and sulfur (S)-oxidizing bacteria (Thiobacillus sp.) increased canola oil production by a maximum of 548 and 335 kg ha−1, respectively. P-solubilizing bacteria (Bacillus sp.) and Thiobacillus sp. enhanced the uptake of different nutrients including nitrogen (N), P, potassium (K), zinc (Zn), and manganese (Mn). Monthly measurements of soil P indicated that soil P fluctuations can be managed using the applied treatments for proper P fertilization in canola production. The results indicated the important role of chemical and biological (Bacillus sp.) P sources and S-oxidizing bacteria for canola growth and oil production as they resulted in significant increase in canola oil production and nutrient uptake. This can be very beneficial for the farmers and industry.