Matricaria chamomilla L. (MC), belonging to the genus Matricaria (Compositae), is an annual plant indigenous to Europe and grows in Xinjiang Uygur Autonomous Region of China, Northern and Western Asia, Europe, and Northern America [1]. The capitula of MC, known as German chamomile, was used to treat some ailments such as digestive ailments, restlessness, mild cases of insomnia due to nervous disorders, and the skin and mucosa inflammation and irritations [2]. Similarly, the whole herb of MC (WHMC) is commonly used as a Uygur herbal medicine for stomach upset, dysuria, skin itching, blurred vision, cystitis, and stomatitis [3]. Previous phytochemical investigations on the capitula of MC have revealed the presence of coumarins, flavonoids, etc. [4–7]. To uncover the bioactive components of WHMC, the phytochemical studies of WHMC were carried out, and 18 phenolic compounds were first isolated and identified from WHMC by us [8]. In our continuing study, 16 compounds, including a neolignan (1), 13 steroids (2–14), and two other constituents (15, 16), were separated from WHMC. Their structures were determined based on spectral data and by comparison of these data with literature values. The isolated compounds were elucidated as dimeric coniferyl isovalerate (1) [9], 7,22-diene-3,5,6-trihydroxyergosterol (2) [10], 7,22-diene-3,5,6-trihydroxyergosterol (3) [11], 3β-hydroxy-5α,8α-epidioxyergosta-6,22-diene (4) [12], 3β-hydroxy- (22E,24R)-ergosta-5,8,22-trien-7-one (5) [13], 6β-hydroxystigmasta-4,22-dien-3-one (6) [14], 6β-hydroxystigmast-4-en-3 one (7) [15], stigmast-22-ene-3,6-dione (8) [16], 5α-stigmasta-3,6-dione (9) [17], 7α-hydroxystigmasterol (10) [18], 7β-hydroxystigmasterol (11) [18], 7α-hydroxysitosterol (12) [19], 7β-hydroxysitosterol (13) [19], β-sitosterol (14), phytol (15), and glyceroyl monopalmitate (16) [20]. Compounds 1–13, 15, and 16 were isolated from MC for the first time, and compounds 1–13, 16 were isolated from the Matricaria genus for the first time. The 13C NMR spectrum of compounds 1 was reported for the first time, and the PMP and 13C NMR spectrum were assigned with the help of 1H–1H COSY, HSQC, and HMBC techniques.