Accessibility of paclitaxel and other taxoids from natural resources is restricted. Endophytic fungi are novel, rapidly growing resources for producing these compounds. Neopestalotiopsis vitis (N. vitis) has been recently isolated from Corylus avellana, and its ability to produce a variety of taxoids has been detected and confirmed by analytical methods. Simultaneous growth and high production of taxoids by application of different sorts and concentrations of carbon and nitrogen were targeted in the present research. These criteria were assessed in different acidities (pH 4.0–7.0), carbon sources (sucrose, fructose, glucose, mannitol, sorbitol, and malt extract), and nitrogen forms (urea, ammonium nitrate, potassium nitrate, ammonium phosphate, and ammonium sulfate) by testing one parameter at a time approach. The first analysis introduced pH 7.0 as the best acidity of the medium for N. vitis, where the highest paclitaxel yield was generated. Further analysis introduced 3% Malt extract as the best carbon-providing medium. In the next step, the effects of nitrogen forms on the growth rate, paclitaxel yield, alkaloids, and amino acid contents were evaluated. Based on the results of this experiment, 5 mM ammonium sulfate was selected as the best nitrogen source to obtain the maximum biomass and paclitaxel yield. Overall, the results introduce a medium containing 3% (w/v) malt extract and 5 mM ammonium sulfate at pH 7.0 as the best medium in which N. vitis produces the highest paclitaxel yield coincident with rapid and sustainable growth. The findings pave the way for industrial manufacturing of taxoids.