Research Highlights: Mycorrhizal helper bacteria (MHB) promote mycorrhization processes and are commonly found in the mycorrhizosphere of fungi, such as the edible hypogeous fungus Tuber melanosporum Vittad. Background and Objectives: The effectiveness of MHB in promoting the mycorrhization process and the root development of Portuguese oak (Quercus faginea Lam.) seedlings destined for truffle plantations has not been determined. The main aim of this study was to shed light on the effect of bacterial inoculation on fungal root tip colonization and seedling root traits. Material and methods: We performed a co-inoculation trial using three bacteria naturally present in the T. melanosporum niche (i.e., Pseudomonas fluorescens, Pseudomonas putida, and Bacillus amyloliquefaciens) and two different bacterial inoculation times (one month and nine months after fungal inoculation) under glasshouse conditions. Results: Only P. fluorescens had a significant mycorrhizal promoter effect, increasing the truffle inoculation rates of root tips by more than 10% compared with seedlings that received non-bacterial inoculation treatments. Simultaneously, the co-inoculation of P. fluorescens with T. melanosporum improved seedling root growth parameters compared with those of seedlings that received non-bacterial inoculation treatments. The different bacterial inoculation times and applications of uninoculated bacterial growth media did not affect the root traits analyzed or the root mycorrhization rates. Conclusions: These results suggest that P. fluorescens bacteria have a potential commercial application as a treatment for truffle-inoculated seedlings to improve both seedling quality and mycorrhizal colonization under nursery conditions.