The chemical composition and antimicrobial activity of the essential oils isolated from twigs with needles (T+N) and from twigs without needles (T-N) from wild Pinus peuce Griseb. (Pinaceae), from three different locations in R. Macedonia, were investigated. Essential oil yields of T+N ranged from 7.5 mL/kg to 12.5 mL/kg and for T-N from 13.8 mL/kg to 17.3 mL/kg. GC/FID/MS analysis of the essential oils revealed eighty-four components, representing 93.7–95.7% and 91.2–92.0% of the T+N and T-N oils, respectively. The major components in T+N and T-N oils were monoterpenes: α-pinene (23.8–39.9%, 21.2–23.3%), camphene (2.2–5.5%, 0.7–2.0%), β-pinene (10.1–17.1%, 8.2–16.4%), myrcene (1.2–1.41%, 1.6–2.5%), limonene+β-phellandrene (6.8–14.0%, 8.8–23.6%) and bornyl acetate (2.3–6.9%, 1.1–3.4%), followed by the sesquiterpenes: trans-(E)-caryophyllene (3.6–4.3%, 3.2–7.3%), germacrene D (7.1–9.5%, 5.0–10.3%) and δ-cadinene (2.1–3.1%, 3.3–4.2%, respectively). Antimicrobial screening of the essential oils was made by disk diffusion and broth dilution methods against 13 bacterial isolates of Gram-positive and Gram-negative bacteria and one strain of Candida albicans. T-N essential oils showed antimicrobial activity toward Streptococcus pneumoniae, Staphylococcus aureus, S. epidermidis and Candida albicans as well as Streptococcus agalactiae, Acinetobacter spp. and Haemophilus influenzae. The antimicrobial activity of T+N essential oils was greater, especially against Streptococcus agalactiae, S. pyogenes, Enterococcus and Candida albicans, followed by Haemophilus influenzae, Acinetobacter spp., Escherichia coli, Salmonella enteritidis, Staphylococcus aureus and S. epidermidis. Minimal inhibitory concentrations (MICs) of all tested essential oils ranged from 15–125 μL/mL. Summarizing the obtained results, the antimicrobial activity of Pinus peuce T+N and T-N essential oils collected from different localities in R. Macedonia varied considerably. These alterations in the antimicrobial activity can be attributed to the differences in the quantitative composition and percentage amounts of the components present in the respective essential oils, although it was evident that there were no differences in the qualitative composition of the essential oils, regardless of the locality of collection, or the type of plant material (T+N or T-N).