Background: Macrophages play a key role in coronary plaque destabilization. In-vitro human monocyte-derived macrophages (MDMs) are used to study macrophages infiltrating tissue. Optical coherence tomography (OCT) provides an in-vivo insight of the coronary arteries. We compared the MDMs morpho-phenotype and culprit plaque features at OCT in acute coronary syndrome (ACS) patients according to the underlying plaque pathobiology. Methods: Sixty-six patients undergoing coronary angiography and pre-angioplasty OCT of the culprit vessel were allocated to three groups according to mechanism of ACS at OCT and C-reactive protein levels (cut-off: 2 mg/Ll): 1) plaque rupture with systemic inflammation; 2) plaque rupture without systemic inflammation, 3) plaque with intact fibrous cap. A blood sample was collected to obtain MDMs, categorized as having “round” or “spindle” morphology. Results: Thirty-two patients (48.5%) were assigned to Group 1, 10 (15.2%) to Group 2 and 24 (36.4%) to Group 3. The “round” MDMs were significantly more frequent in Group 1 (39.25 ± 4.98%) than in Group 2 (23.89 ± 3.10%) and Group 3 (23.02 ± 7.89%), p = 0.008. MDMs in Group 1 as compared to Groups 2 and 3 showed lower efferocytosis (8.74 ± 1.38 vs 9.74 ± 2.15 vs 11.41 ± 2.41; p = 0.012), higher tissue factor levels (369.84 ± 101.13 vs 301.89 ± 59.78 vs 231.74 ± 111.47; p = 0.001) and higher heme oxygenase-1 expression (678.78 ± 145.43 vs 419.12 ± 74.44 vs 409.78 ± 64.33; p = 0.008). Conclusions: MDMs of ACS patients show morpho-phenotypic heterogeneity with prevalence of pro-thrombotic and pro-oxidative properties in case of plaque rupture and systemic inflammation. Such MDMs subpopulation may take part to the cellular pathways leading to fibrous cap rupture with the subsequent thrombus formation.