Conventional magnetic resonance imaging (MRI) often acquires no signal in anterior cruciate ligament (ACL) due to the short apparent transverse relaxation time of ACL. Ultrashort echo time (UTE) MRI is capable of imaging ACL with high signal which enables quantitative ACL assessment. This study aimed to investigate the correlations of the mechanical and microstructural properties of human ACL specimens with quantitative three-dimensional UTE Cones (3D-UTE-Cones) MRI measures. ACL specimens were harvested from cadaveric knee joints of 13 (50.9 ± 21.1 years old, 11 males and 2 female) donors. Specimens were scanned using a series of quantitative 3D-UTE-Cones T₂^* (UTE-T₂^*), T₁ (UTE-T₁), Adiabatic T_1ρ (UTE-Adiab-T_1ρ), and magnetization transfer (UTE-MT) sequences in a wrist coil on a clinical 3T scanner. ACL elastic modulus was measured using a uniaxial tensile mechanical test. Histomorphometry analysis was performed to measure the average fascicle specific surface, fascicle size, and number of cells per unit area. Spearman's rank correlations of UTE-MRI biomarkers with mechanical and histomorphometry measures were investigated. The elastic modulus of ACL showed significant moderate correlations with UTE-Adiab-T_1ρ (R = −0.59, p = 0.01), macromolecular fraction from MT modeling (R = 0.54, p = 0.01), magnetization transfer ratio (R = 0.53, p = 0.01), UTE-T₂^* (R = −0.53, p = 0.01), and average fascicle specific surface (R = 0.54, p = 0.01). UTE-MRI showed nonsignificant correlations with histomorphometry measures. UTE-MRI biomarkers may be useful noninvasive tools for the ACL mechanical assessment.