During the last decade, the application of pretreatment has been investigated to enhance methane production from lignocellulosic biomass such as wheat straw (WS). Nonetheless, most of these studies were conducted in laboratory batch tests, potentially hiding instability problems or inhibition, which may fail in truly predicting full-scale reactor performance. For this purpose, the effect of an alkaline pretreatment on process performance and methane yields from WS (0.10 g NaOH g−1 WS at 90 °C for 1 h) co-digested with fresh wastewater sludge was evaluated in a pilot-scale reactor (20 L). Results showed that alkaline pretreatment resulted in better delignification (44%) and hemicellulose solubilization (62%) compared to untreated WS. Pilot-scale study showed that the alkaline pretreatment improved the methane production (261 ± 3 Nm3 CH4 t−1 VS) compared to untreated WS (201 ± 6 Nm3 CH4 t−1 VS). Stable process without any inhibition was observed and a high alkalinity was maintained in the reactor due to the NaOH used for pretreatment. The study thus confirms that alkaline pretreatment is a promising technology for full-scale application and could improve the overall economic benefits for biogas plant at 24 EUR t−1 VS treated, improve the energy recovery per unit organic matter, reduce the digestate volume and its disposal costs.