In recent years, it has been suggested that a potential source of nitrogen for conifer trees growing in nitrogen-limited boreal forests could be nitrogen fixation by endophytic diazotrophic bacteria inhabiting coniferous needles. In fact, nitrogen-fixing bacteria have been isolated from needles of diverse conifer species, like white spruce, lodgepole pine, and western redcedar. For some conifer species, active nitrogen fixation has been reported. Our study assessed whether nitrogen-fixing bacteria are also present and active in the needles of Scots pine in control and inorganic nitrogen-fertilized plots within the Swedish boreal forest. Western blot was used to look at the presence of the nifH subunit of enzyme nitrogenase, which is responsible for nitrogen fixation, and acetylene-reduction assay was used to measure its activity. Interestingly, both the presence and activity of bacteria were similar between the control and inorganic nitrogen fertilized plots. Nitrogen-fixing bacteria were isolated using nitrogen-free media, and these bacteria belonged to Bacillus, Variovorax, Microbacterium, Sphingomonas, Novosphingobium, and Priestia genera. The nitrogen fixation ability of these bacteria was confirmed by in vitro measurements of acetylene reduction. Additionally, we assessed whether the isolated bacteria possess any other plant growth-promoting properties, such as phosphorus and zinc solubilization, siderophore, hydrogen cyanide, and indole-3-acetic acid production, using in vitro assays.